Availability of proteins that specifically bind or interact with target proteins or other molecules has for some time been of importance in biology and medicine. For example, medical diagnosis has been revolutionized by assays using high-affinity proteins, mainly, antibodies, that bind to disease markers. High-affinity antibodies to disease-causing agents are of increasing importance in medical therapeutics. In biological research, high affinity proteins, also mainly antibodies, have found use in the purification of rare proteins, in the localization of proteins or other antigens in cells such as by immuno-histochemical techniques, and in countless other applications. High-affinity proteins are likely to assume increasing importance in the future (Binz et al., 2005, Nat Biotechnol, 23: 1257-68.). For example, the emerging field of proteomics seeks to understand the patterns of expression and interaction of a substantial fraction of the proteins encoded in a cell's genome.
However, existing methods of providing binding proteins or polypeptides that bind with affinity and specificity to selected targets, especially to large numbers of selected targets, has been and continues to be difficult and expensive. The predominant method used today is to raise antibodies, either monoclonal or polyclonal, against a target molecule. Although well known and widely used, this strategy has several limitations and disadvantages (Binz et al., 2005, Nat Biotechnol, 23: 1257-68.). First, to generate, or “raise”, an antibody against a target requires either a sufficient amount of the purified target itself or a chemically synthesized fragment of the target. Second, raising an antibody normally requires the use of living animals, and due to species incompatibilities, it is not always possible to raise a specific antibody against a particular target, much less against large numbers of targets, such as a significant fraction of the proteins in an organism. Third, isolation and production of antibodies are expensive, time-consuming and unpredictable processes. Fourth, antibodies cannot be expressed via recombinant hosts without significant investment of time and money because the antigen-binding regions of the antibody heavy and light chains must be cloned, sequenced, and then simultaneously expressed. Finally, antibodies usually do not fold properly in the reductive cell environment, and therefore are not useful to target intracellular molecules involved in disease. Such limitations and disadvantages constitute a significant barrier to the rapid identification, diagnosis and treatment of infectious diseases such as AIDS, SARS, West Nile virus, and anthrax, or of non-infectious diseases such as cancer.
An alternative method relies on “directed evolution” to alter the binding specificity of naturally-occurring proteins that are known to bind to determined targets. In this method, a known gene is randomly mutated by a chemical or biotechnological mutagenesis technique, for example, by PCR-based mutagenesis, or by insertion of randomized codons in regions corresponding to elements of protein secondary structure called “loops”, e.g., (Legendre et al., 2002, Protein Sci, 11: 1506-18.), or by randomization of codons that normally constitute a loop (e.g., Xu et al., 2002, Chem Biol, 9: 933), and other references reviewed in (Binz et al., 2005, Nat Biotechnol, 23: 1257-68.). Then a library of the resulting protein variants is screened for variants having affinity to a new target, for example, by phage display. In this way, several proteins have been “evolved” in the laboratory to create protein variants having useful new specificities, e.g., (Xu et al., 2002, Chem Biol, 9: 933), and reviewed in (Binz et al., 2005, Nat Biotechnol, 23: 1257-68.). Alternatively, “loop grafting” wherein an insertion of a preexisting amino acid chain that is known to bind a target molecule of interest is made in one loop of a “scaffold protein”, as was done by Nicaise and colleagues (Nicaise et al., 2004, Protein Sci, 13: 1882-91. Epub 2004 May 28.), can also be carried out to confer a novel binding specificity to the scaffold protein.
A further alternative is to create novel binding proteins de novo through directed evolution. However, proteins having no natural counterparts, e.g., iMabs from Catchmabs BV or as described by (Keefe et al., 2001, Nature, 410: 715-8), have significant drawbacks such as, for example, that they are likely to be recognized as foreign by the human immune system, thereby impeding their use as therapeutics. For the same reason, natural proteins of non-human origin engineered to bind target polypeptides (e.g., Ronnmark et al., 2002, Eur J Biochem, 269: 2647-55.; Zeytun et al., 2003, Nat Biotechnol, 21: 1473-9.) are unlikely to be useful as therapeutics or diagnostics.
Thus, the choice of binding protein to be modified via directed evolution will strongly influence the utility of the evolved binding proteins. PDZ domains constitute an example of a family of binding proteins which can be used to create novel research reagents (Ferrer et al., 2005, Protein Eng Des Sel, 18: 165-173), diagnostic reagents or therapeutics having many advantages over existing binding proteins (WO 2005/072159). Such advantages include ease and speed of isolation using in vitro methods, low cost of production using non-mammalian host cells, potential utility as intracellular biotherapeutics due to their natural propensity to function in the cytoplasm, and lack of immunogenicity.
PDZ domains are relatively well understood and of great potential utility. They participate in signal transduction pathways by mediating protein complex formation and are also involved in targeting of proteins to various locations within the cell. In metazoan genomes, including the human genome, PDZ domains are among the most common protein sequence modules. Recent reviews on PDZ domains include refs. (Hung et al., 2002, J Biol Chem, 277: 5699-702) and (Fan et al., 2002, Neurosignals, 11: 315-21). Many PDZ domains are stable and expressed to high levels in recombinant bacterial hosts, which has facilitated their extensive biophysical characterization (e.g., Morais Cabral et al., 1996, Nature, 382: 649-52.; Cohen et al., 1998, J Cell Biol, 142: 129-38.; Daniels et al., 1998, Nat Struct Biol, 5: 317-25.; Im et al., 2003, J Biol Chem, 278: 8501-7). PDZ domains have been described as potential therapeutics, for example to treat cancer by interfering with Myc protein function. See for example, (Junqueira et al., 2003, Oncogene, 22: 2772-81) and US Pat. App. Pub. No. 20030119716. Other PDZ patent applications expand the utility of PDZ domains by describing engineered PDZ domain fusions, or chimeras, with other proteins (US Patent Application Pub. Nos. 20010044135, 20020037999, and 20020160424). PDZ domains can also be used to identify drug candidates in high-throughput screens (Ferrer et al., 2002, Anal Biochem, 301: 207-16; Hamilton et al., 2003, Protein Sci, 12: 458-67) (Ferrer et al., 2005, Protein Eng Des Sel, 18: 165-173).
Some progress has been made in studying and modifying the binding specificity of PDZ domains. Schneider et al., 1999, Nature Biotechnology 17:170-175 and (Junqueira et al., 2003, Oncogene, 22: 2772-81) both describe how the binding specificity of a naturally-occurring PDZ domain can be altered using directed evolution methods. Phage display may be used to determine the specificity of a given PDZ domain (see, e.g., Fuh et al., 2000, J. Biol. Chem. 275:21486-91). In this work, Fuh and colleagues selected phage-displayed random C-terminal peptide sequences capable of binding to an immobilized PDZ domain. However, this approach is not intended to, and cannot alter the specificity of a given PDZ domain. Skelton et al. (2003, J. Biol. Chem., 278: 7645-54), propose the use of phage display to alter PDZ domain specificity, but the paper by Ferrer et al. (Ferrer et al., 2005, Protein Eng Des Sel, 18: 165-173) is the first experimental demonstration of this concept. Phage display is believed to provide greater control over the conditions of the binding interactions, including affinity and specificity, than is afforded by two-hybrid selections which are notoriously artifact-prone.
Alternatively, PDZ domains with altered binding specificity may be designed by computational methods, as shown by (Reina et al., 2002, Nat Struct Biol, 9: 621-7) and US Patent Application Pub. No. 20030059827. These computational methods seem to offer several apparent benefits, such as reduced cost and time by avoiding experimental effort, and scalability for determining binding proteins to multiple targets. On the other hand, these methods have certain notable drawbacks such as the well-known extreme difficulty of predicting binding affinities of designed protein structures, yielding candidate binding proteins of unreliable affinity and specificity. Also, once structures have been designed in silico, the corresponding proteins must still be prepared in the laboratory. The effort required to construct the candidate gene variants is similar to the effort required to prepare a library of mutant genes, and once such a library is constructed, it can be screened multiple times with diverse targets whereas new variants must be designed and synthesized for each new target. Finally, design of variant binding proteins and optimization of their binding affinity is extremely difficult without the availability of detailed information on their atomic structure, while directed evolution has no such need. The acquisition of this type structural data is costly and slow, often requiring months of work.
To fully realize the potential of PDZ domains as diagnostics and therapeutics, the ability to engineer alternative or additional binding sites in PDZ domains is highly desirable. The majority of PDZ domains that have been studied so far bind to the last few (three to seven) residues at the carboxyl terminus of their cognate polypeptide ligands. However, because a significant fraction of molecular targets have inaccessible carboxyl termini or termini that do not participate in disease mechanisms, PDZ variants capable of binding other features of target proteins would have desirable versatility and utility as research reagents, diagnostic reagents, and therapeutics.
In summary, polypeptides capable of binding to specific targets, especially to natural peptide sequences, are useful in biology and medicine, and are expected to be of increasing utility in the future. Therefore, inexpensive and efficient methods for providing diverse binding proteins capable of functioning as affinity reagents and/or therapeutics are needed.